The present invention relates to methods for administering gene products to animals using adenoviral vectors.
Modified viruses have proven convenient vector systems for investigative and therapeutic gene transfer applications, and adenoviral vector systems present several advantages for such uses. Adenoviruses are generally associated with benign pathologies in humans, and the 36 kb of the adenoviral genome has been extensively studied. Adenoviral vectors can be produced in high titers (e.g., about 1013 pfu), and such vectors can transfer genetic material to nonreplicating, as well as replicating, cells; in contrast with, e.g., retroviral vectors, which only transfer genetic material to replicating cells. The adenoviral genome can be manipulated to carry a large amount of exogenous DNA (up to about 8 kb), and the adenoviral capsid can potentiate the transfer of even longer sequences (Curiel et al., Hum. Gene Ther. 3: 147-154 (1992)). Additionally, adenoviruses generally do not integrate into the host cell chromosome, but rather are maintained as a linear episome, thus minimizing the likelihood that a recombinant adenovirus will interfere with normal cell function. Aside from being a superior vehicle for transferring genetic material to a wide variety of cell types, adenoviral vectors represent a safe choice for gene transfer, a particular concern for therapeutic applications.
A variety of recombinant adenoviral vectors have been described. Most of the vectors in use today derive from the adenovirus serotype 5 (Ad5), a member of subgroup C. An exogenous gene of interest typically is inserted into the early region 1 (E1) of the adenovirus. Disruption of the E1 region decreases the amount of viral proteins produced by both of the early regions (DNA binding protein) and late regions (penton, hexon, and fiber proteins), preventing viral propagation. Replication-deficient adenoviral vectors require growth in either a complementary cell line or in the presence of an intact helper virus, which provides, in trans, the essential E1 functions (Berker et al., J. Virol. 61: 1213-1220 (1987); Davidson et al., J. Virol. 61: 1226-1239 (1987); Mansour et al., Mol. Cell Biol. 6: 2684-2694 (1986)). More recently, adenoviral vectors deficient in both E1 and the early region 4 (E4) have been used to substantially abolish expression of viral proteins. In order to insert the larger genes (up to 8 kb) into the adenoviral genome, adenoviral vectors additionally deficient in the nonessential early region 3 (E3) and the early region 2 (E2) can be used. Multiply deficient adenoviral vectors are described in published PCT patent application WO 95/34671.
One limitation of adenoviral vector systems is the ability of the adenoviral vector to transduce a wide variety of proliferating and quiescent cells (Michou et al., Gene Ther. 4: 473-482 (1997)). This ability, while a benefit in transducing the target area, is a limitation when the adenoviral vector xe2x80x9cleaksxe2x80x9d out of the targeted area and transduces other cells it contacts. Tranduction of the surrounding cells is a serious problem when the gene product encoded by the adenoviral vector is harmful, toxic, or otherwise undesirable with respect to these non-targeted areas.
Another limitation of the adenoviral vector system is the cellular and humoral immune response generated within the host animal. Initial administration elicits a reaction from both CD8+ and CD4+ T lymphocytes, which eliminate virus infected cells within 28 days after infection, limiting the duration of the transgene expression. In addition, neutralizing antibodies produced by B lymphocytes in cooperation with CD4+ cells inhibit the effectiveness of repeat administration of the adenoviral vector. Proliferation and specificity of the antibodies to the adenoviral vectors occurs through interactions among the adenoviral vector, B-cell surface immunoglobulins and activated CD4+ surface proteins (particularly CD40 ligand (CD40L), which binds CD40 on the surface of B cells) (Yang et al., J. Virol. 69: 2004 (1995)).
Attempts to circumvent the humoral immune response to allow repeat administration of the adenoviral vector have met with limited success. These attempts have focused in two areas: immunosuppression and alteration of the adenoviral vector. Several groups have experimented with various immunosuppressant drugs or antibodies specific for CD4+, CD40L, or CTLA4Ig to reduce the adenovirus-specific humoral immune response (Lee et al., Hum. Gene Ther. 7: 2273 (1996) (CD4+ ); Yang et al., J. Virol. 70: 6370 (1996) (CD40L); Kay et al., Nature Gen. 11: 191 (1995) (CTLA4Ig)). Although some of these results have been encouraging, there is a substantial risk associated with systemic immune suppression in a clinical setting. Alteration of the adenoviral vector is time consuming and has not been entirely successful in sufficiently attenuating the immune response. Limited readministration of the adenoviral vector has been accomplished when adenoviral vectors of different serotypes within the same subgroup are used; however, persistence of expression of the transgene was not comparable to the initial administration (Mack et al., Hum. Gene Ther. 8: 99-109 (1997)).
Accordingly, there is a need for improved methods of administering adenoviral vectors to animals, particularly, to prevent leakage of the adenoviral vector from the target area and to circumvent the humoral immune response elicited by adenoviral vectors. The present invention provides such methods. This and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention provides a method of targeting a gene product to a particular muscle of an animal. The method comprises inducing in an animal systemic neutralizing antibodies to an adenoviral gene transfer vector and administering the adenoviral gene transfer vector comprising an exogenous gene encoding a gene product to a particular muscle of the animal. Administration is such that the exogenous gene is expressed and the gene product is produced in the particular muscle of the animal and the adenoviral gene transfer vector is neutralized outside of the particular muscle of the animal by the systemic neutralizing antibodies.
The present invention further provides a method of producing a gene product in a skeletal muscle of an animal. The method comprises initially administering an adenoviral vector to a skeletal muscle of an animal, and, at least seven days after administration, subsequently administering an adenoviral gene transfer vector comprising an exogenous gene encoding a gene product to the skeletal muscle of the animal. Administration is such that the exogenous gene is expressed and the gene product is produced in the skeletal muscle of the animal.
The invention may best be understood with reference to the following detailed description.
The present invention provides methods useful in the administration of gene products to animals using adenoviral gene transfer vectors. The ability to target an adenoviral vector and to administer repeatedly a therapeutic adenoviral vector in a clinical setting is useful in improving treatment efficacy and in enabling the treatment of diseases. This invention provides a method to limit the infection of non-target tissue following administration of an adenoviral vector to a particular muscle of an animal. The vector targeting potential is useful for cardiac, particularly, endocardial, administration, as the risk of misinjection of the adenoviral vector is high. As adenoviral vectors cannot be readministered systemically, the present invention also provides a method for repeat administration of an adenoviral gene transfer vector comprising an exogenous gene to the skeletal muscle of an animal.
The term xe2x80x9cexogenous gene,xe2x80x9d as it is used herein, refers to any gene in an adenoviral gene transfer vector that is not native to the adenovirus that comprises the adenoviral vector. The gene includes a nucleic acid sequence encoding a gene product operably linked to a promoter. Any portion of the gene can be non-native to the adenovirus that comprises the adenoviral gene transfer vector. For example, the gene can comprise a non-native nucleic acid sequence encoding a gene product operably linked to a native promoter, or a native nucleic acid sequence encoding a gene product operably linked to a non-native promoter or in a non-native location within the adenoviral vector. It should be appreciated that the exogenous gene can be any gene encoding an RNA or protein of interest to the skilled artisan. Therapeutic genes, genes encoding a protein that is to be studied in vitro and/or in vivo, antisense nucleic acids, and modified viral genes are illustrative of possible exogenous genes.
The term xe2x80x9cadenoviral gene transfer vector,xe2x80x9d as it is used herein, refers to any adenoviral vector with an exogenous gene encoding a gene product inserted into its genome. The vector must be capable of replicating and being packaged when any deficient essential genes are provided in trans. An adenoviral vector desirably contains at least a portion of each terminal repeat required to support the replication of the viral DNA, preferably at least about 90% of the full ITR sequence, and the DNA required to encapsidate the genome into a viral capsid. Many suitable adenoviral vectors have been described in the art.
The adenoviral gene transfer vector is preferably deficient in at least one gene function required for viral replication. Preferably, the adenoviral gene transfer vector is deficient in at least one essential gene function of the E1 region of the adenoviral genome, particularly the E1 a region, more preferably, the vector is deficient in at least one essential gene function of the E1 region and part of the E3 region (e.g., an Xba I deletion of the E3 region) or, alternatively, the vector is deficient in at least one essential gene function of the E1 region and at least one essential gene function of the E4 region. However, adenoviral gene transfer vectors deficient in at least one essential gene function of the E2a region and adenoviral gene transfer vectors deficient in all of the E3 region also are contemplated here and are well-known in the art. Suitable replication-deficient adenoviral gene transfer vectors are disclosed in International Patent Applications WO 95/34671 and WO 97/21826. For example, suitable replication-deficient adenoviral gene transfer vectors include those with a partial deletion of the E1a region, a partial deletion of the E1b region, a partial deletion of the E2a region, and a partial deletion of the E3 region. Alternatively, the replication-deficient adenoviral gene transfer vector can have a deletion of the E1 region, a partial deletion of the E3 region, and a partial deletion of the E4 region.
It should be appreciated that the deletion of different regions of the adenoviral gene transfer vector can alter the immune response of the mammal, in particular, deletion of different regions can reduce the inflammatory response generated by the adenoviral gene transfer vector. Furthermore, the adenoviral gene transfer vector""s coat protein can be modified so as to decrease the adenoviral gene transfer vector""s ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509. Other suitable modifications to the adenoviral gene transfer vector are described in U.S. Pat. Nos. 5,559,099; 5,731,190; 5,712,136; and 5,846,782 and International Patent Applications WO 97/20051, WO 98/07877, and WO 98/54346.
Adenoviral gene transfer vectors can be specifically targeted through a chimeric adenovirus coat protein comprising a nonnative amino acid sequence, wherein the chimeric adenovirus coat protein directs entry into a specific cell of an adenoviral gene transfer vector comprising the chimeric adenovirus coat protein that is more efficient than entry into a specific cell of an adenoviral gene transfer vector that is identical except for comprising a wild-type adenovirus coat protein rather than the chimeric adenovirus coat protein. The chimeric adenovirus coat protein comprising a nonnative amino acid sequence can serve to increase efficiency by decreasing non-target cell transduction by the adenoviral gene transfer vector. The nonnative amino acid sequence of the chimeric adenovirus coat protein, which comprises from about 3 amino acids to about 30 amino acids, can be inserted into or in place of an internal coat protein sequence, or, alternatively, the nonnative amino acid sequence can be at or near the C-terminus of the chimeric adenovirus coat protein. The chimeric adenovirus coat protein can be a fiber protein, a penton base protein, or a hexon protein. In addition, the nonnative amino acid sequence can be linked to the chimeric adenovirus coat protein by a spacer sequence of from about 3 amino acids to about 30 amino acids. Targeting through a chimeric adenovirus coat protein is described generally in U.S. Pat. Nos. 5,559,099; 5,712,136; 5,731,190; 5,770,440; 5,871,726; and 5,830,686 and International Patent Applications WO 96/07734, WO 98/07877, WO 97/07865, WO 98/54346, WO 96/26281, and WO 98/40509. An adenoviral gene transfer vector that comprises a chimeric coat protein comprising a nonnative amino acid sequence in accordance with U.S. Pat. No. 5,965,541 or WO 97/20051, such as one that comprises polylysine as the nonnative amino acid sequence, can be used to re-administer an exogenous gene encoding a gene product to a particular muscle of an animal. The use of such a vector to repeat administration can result in a higher level of expression of the gene product as compared to an adenoviral vector in which the corresponding adenoviral coat protein has not been modified to comprise a nonnative amino acid sequence, such as polylysine.
The exogenous gene can be inserted into any suitable region of the adenoviral gene transfer vector as an expression cassette. Preferably, the DNA segment is inserted into the E1 region of the adenoviral gene transfer vector. Whereas the DNA segment can be inserted as an expression cassette in any suitable orientation in any suitable region of the adenoviral gene transfer vector, preferably, the orientation of the DNA segment is from right to left. By the expression cassette having an orientation from right to left, it is meant that the direction of transcription of the expression cassette is opposite that of the region of the adenoviral gene transfer vector into which the expression cassette is inserted.
In one embodiment, the present invention provides a method of targeting a gene product to a particular muscle of an animal. The method comprises inducing in an animal systemic neutralizing antibodies to an adenoviral gene transfer vector and then administering the adenoviral gene transfer vector comprising an exogenous gene encoding a gene product to a particular muscle of the animal. Administration is such that the exogenous gene is expressed and the gene product is produced in the particular muscle of the animal and the adenoviral gene transfer vector is neutralized outside of the particular muscle of the animal.
The present invention can be practiced with any suitable animal, preferably the present invention is practiced with a mammal, more preferably, a human. Additionally, the adenoviral gene transfer vector can be administered to any suitable muscle of the animal.
Any suitable method can be used to induce systemic neutralizing antibodies to the adenoviral gene transfer vector. Desirably, an antigen is administered to the animal to produce systemic neutralizing antibodies to the adenoviral gene transfer vector. This antigen can be the same as the adenoviral gene transfer vector, but preferably, it is the same as the adenoviral gene transfer vector, except that it does not contain an exogenous gene (i.e., a null vector). The antigen also can be administered by any suitable method. Depending on the antigen, administration can be to any suitable area of the animal. In order to induce the systemic neutralizing antibodies, the antigen can be administered any number of suitable times, e.g., once, twice, or more.
Using a null vector, the antigen can be administered systemically (rather than to the target muscle) to prevent any damage to the particular muscle. Systemic administration can be accomplished through intravenous injection, either bolus or continuous, or any other suitable method.
Administration of the antigen produces circulating neutralizing antibodies. While not wishing to be bound by any particular theory, it is believed that when the adenoviral gene transfer vector is administered to the particular muscle of the animal, some of the adenoviral particles escape the muscle. These adenoviral particles are then neutralized by the antibodies circulating throughout the animal such that significantly less (and preferably substantially no) gene product is produced outside the particular muscle. The amount of gene product produced outside the particular muscle of administration in the animal is preferably at least 90% less (more preferably at least 99% less, and most preferably at least 99.9% less) than the production of the gene product outside the particular muscle of administration in a naive animal of the same species as the animal after administration of the adenoviral gene transfer vector comprising an exogenous gene. A naive animal is one that does not have circulating neutralizing antibodies to the adenoviral gene transfer vector.
Methods are known in the art for comparing the amount of gene product outside the muscle that is the site of administration in an animal with systemic neutralizing antibodies with the amount of gene product outside the same muscle that is the site of administration in a naive animal. For example, the comparison can be made at the same time after administration of the adenoviral gene transfer vector and between the same sites of the two animals.
Neutralization of adenoviral particles outside of the particular muscle prevents production of the exogenous gene carried in the adenoviral gene transfer vector. This is extremely useful in situations where the exogenous gene is harmful, or toxic, to the animal when present in areas other than the particular muscle of administration. An example of this is vascular endothelial growth factor (VEGF protein), which mediates vascular growth. While vascular growth is desirable in the heart to repair damaged cardiac muscle, growth outside the heart can lead to severe problems, including blindness and increased aggressiveness of tumor cells.
In view of the above, the method can further comprise subsequently repeating the administration of an adenoviral gene transfer vector comprising the exogenous gene encoding the gene product to the particular muscle of the animal. When the administration is repeated, the adenoviral gene transfer vector comprising the exogenous gene encoding the gene product preferably further comprises a chimeric adenoviral coat protein comprising a nonnative amino acid sequence, wherein the chimeric adenoviral coat protein directs entry of the vector into cells more efficiently than a vector that is otherwise identical, except for comprising a corresponding wild-type adenoviral coat protein (see, e.g., U.S. Pat. No. 5,965,541 or WO 97/20051). Preferably, the nonnative amino acid sequence consists essentially of polylysine, such as from about 3 to about 30 lysines.
In another embodiment, the present invention provides a method of producing a gene product in a skeletal muscle of an animal. The method comprises initially administering an adenoviral vector to a skeletal muscle of an animal, and, at least seven days after, subsequently administering an adenoviral gene transfer vector comprising an exogenous gene encoding a gene product to the skeletal muscle of the animal. Administration is such that the exogenous gene is expressed and the gene product is produced in the skeletal muscle of the animal.
Any suitable animal can be used; however, preferably, the animal is a mammal, more preferably, a human. In the context of the present invention, the adenoviral vector initially administered to the skeletal muscle of the animal can be the same as, or different from, the adenoviral gene transfer vector comprising an exogenous gene encoding a gene product subsequently administered at least seven days after the initial administration.
After subsequent administration of the adenoviral gene transfer vector comprising an exogenous gene, production of the gene product in the muscle of the animal is desirably at least 1% of (such as at least 10% of, preferably at least 50% of, more preferably at least 80% of, and most preferably, the same as or substantially the same as) production of the gene product after initial administration of the same adenoviral gene transfer vector containing the exogenous gene. Methods for comparing the amount of gene product produced in the muscle of administration are known in the art. The comparison can be made at the same time after the initial and subsequent administrations of the adenoviral gene transfer vector.
While not wishing to be bound by any particular theory, it is believed that the level of gene product produced in the skeletal muscle of an animal after the second or subsequent administration to the muscle can be substantially similar to that of the first or preceding administration because neutralizing antibodies, which are produced by the first or preceding administration, cannot readily penetrate the muscle and destroy the adenoviral gene transfer vector. When the neutralizing antibody response is boosted with two or more initial administrations of the adenoviral vector before the subsequent administration of the adenoviral gene transfer vector comprising the exogenous gene, the level of gene product produced in the skeletal muscle of administration may be lowered, yet still sufficient to produce a therapeutic or prophylactic effect.
In view of the above, the method can further comprise subsequently repeating the administration of an adenoviral gene transfer vector comprising the exogenous gene encoding the gene product to the skeletal muscle of the animal. When the administration is repeated, the adenoviral gene transfer vector comprising the exogenous gene encoding the gene product preferably further comprises a chimeric adenoviral coat protein comprising a nonnative amino acid sequence, wherein the chimeric adenoviral coat protein directs entry of the vector into cells more efficiently than a vector that is otherwise identical, except for comprising a corresponding wild-type adenoviral coat protein (see, e.g., U.S. Pat. No. 5,965,541 or WO 97/20051). Preferably, the nonnative amino acid sequence consists essentially of polylysine, such as from about 3 to about 30 lysines.
To facilitate the administration of adenoviral vectors, they can be formulated into suitable pharmaceutical compositions. Generally, such compositions include the active ingredient (i.e., the adenoviral vector) and a pharmacologically acceptable carrier. Such compositions can be suitable for delivery of the active ingredient to a patient for medical application, and can be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more pharmacologically or physiologically acceptable carriers comprising excipients, as well as optional auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Thus, for injection, the active ingredient can be formulated in aqueous solutions, preferably in physiologically compatible buffers. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the active ingredient can be combined with carriers suitable for inclusion into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For administration by inhalation, the active ingredient is conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant. The active ingredient can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Such compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Other pharmacological excipients are known in the art.
Those of ordinary skill in the art can easily make a determination of the proper dosage of the adenoviral gene transfer vector. Generally, certain factors will impact the dosage that is administered; although the proper dosage is such that, in one context, the exogenous gene is expressed and the gene product is produced in the particular muscle of the mammal. Preferably, the dosage is sufficient to have a therapeutic and/or prophylactic effect on the animal. The dosage also will vary depending upon the exogenous gene to be administered. Specifically, the dosage will vary depending upon the particular muscle of administration, including the specific adenoviral vector, exogenous gene and/or promoter utilized. For purposes of considering the dose in terms of particle units (pu), also referred to as viral particles, it can be assumed that there are 100 particles per particle forming unit (pfu) (e.g., 1xc3x971012 pfu is equivalent to 1xc3x971014 pu).
The present inventive methods are useful in the context of the treatment of animals, e.g., medical treatment. In addition, the present inventive methods are useful in the production of gene products, e.g., in vivo protein production (which can entail subsequent protein recovery) as well as in research, e.g., investigation of gene expression, adenoviral targeting, and the like.